Gates-Baby-BCG-Panel 2 files

These samples are part of a Baby BCG study funded by the Gates Foundation. The assay type is EpiTOF (surface stain and intracellular epigenetic marker staining) of unstimulated cryopreserved cells. There are two timepoints per donor. There are Adolescent and Baby PBMCs, and Cord Blood. For each Plate, the surface antibody staining was performed as separate samples, then the samples were fixed with PFA, permed with Methanol, and barcoded using the Fluidigm/Standard Biotools 20plex Pd kit. The samples were then Pooled, and intracellular antibody and Ir staining was performed. EQ4 beads were added, and the sample was acquired in MilliQ water and with the NB injector. The Pooled sample was acquired on the Helios mass cytometer, then the files were normalized (Passport EQ-P13H2302_ver2), concatenated, and debarcoded using the Helios software. There were two different intracellular cocktails used: Panel 1 (primarily acetylation markers) and Panel 2 (primarily methylation markers). After barcoding, the sample was counted on a Bio-Rad TC20 cell counter. If more than 10M cells were recovered, the sample was split in half and stained separately with each panel. If fewer than 10M cells were recovered, the sample was not split and only Panel 1 was stained. Surface cocktail antibodies were prepared fresh each day. Intracellular antibody cocktails were composed of 2 parts: a frozen cocktail and a fresh cocktail (not all antibodies could retain full staining after freezing). See Excel cocktail sheet for more details. The Helios acquisition template in both cases was the same. For Panel 1, the relevant marker is the first in the channel name (prior to the "-"). For Panel 2, the relevant marker is the second in the channel name (after the "-")
Notes:
This file set is the Panel 2 set. These FCS files are Fluidigm/Sbio-normalized, debarcoded, Ungated. They still contain Beads, debris, etc. The corresponding Panel 1 files can be found at FR-FCM-Z8FS EQ4 commercial normalization beads were included during sample running. After acquisition, the files were normalized on the Helios software. The 1217 PBMC donor was used as a QC staining control on each plate.